My laboratory focuses on the functional analysis of the human breast cancer susceptibility genes, BRCA1 and BRCA2 . Breast cancer is the most frequently diagnosed cancer in women in the United States. It has been estimated that about 178,480 new cases of invasive breast cancer were diagnosed and more than 40,400 individuals died from this disease in 2007. Among the various factors responsible for the development of this cancer, a family history of the disease seems to play a major role. Mutations in BRCA1 and BRCA2 are linked to increased risk of early onset familial breast and ovarian cancers. Individuals with mutations in either of these genes are also at risk for developing cancer in other organs as well. The penetrance of the disease in BRCA1 and BRCA2 mutation carriers has been estimated to be 35-80%. In an effort to reduce the mortality from breast cancer through prevention and early diagnosis, BRCA1 and BRCA2 mutation carriers are encouraged to undergo intensive screening and, in some cases, prophylactic surgery or chemoprevention. Sequencing based genetic tests are available to identify BRCA1 and BRCA2 mutation carriers. A major caveat of this approach lies in the interpretation of the actual risk(s) associated with mutations such as single nucleotide substitutions, small in-frame deletions or insertions, or splice-site mutations that do not obviously disrupt the gene product. The need to determine the functional significance of such sequence variants is fast growing because of the given increase in the number of people being assessed for mutation in BRCA1 and BRCA2 and because of the potential for invasive outcomes, it will be essential to discriminate among mutations with respect to the severity of disease. Currently, association analyses in families are used to determine whether a mutation poses a risk. While data from epidemiological studies would be valuable in assessing the cancer risk for each particular mutation, they have identified a limited number of variants with clear association. Consequently, the vast majority of such variants are listed as unclassified variants in the Breast Cancer Information Core (BIC) database. To evaluate the functional significance of human BRCA1 and BRCA2 variants and to carry out functional dissection of these proteins, we have developed a simple, reliable and physiologically relevant functional assay using mouse embryonic stem (ES) cells. The assay is based on the fact that Brca and Brca2 -null ES cells cannot survive. Therefore we engineered ES cells to express a conditional allele of Brca1 or Brca2. Desired mutations are generated in the human BRCA1 or BRCA2 gene in bacterial artificial chromosomes (BAC) and introduced into the ES cells. Functional analysis is performed after deletion of the conditional allele. The ability of and the extent to which specific variants complement the lethality of ES cells associated with Brca1 or Brca2 deficiency is used to evaluate their functional significance. Variants that can rescue lethality are then screened for a defect in the known functions of BRCA1 and BRCA2. To examine the physiological significance of potentially deleterious human variants, we are generating humanized mouse models by BAC transgenesis. BRCA1 or BRCA2 mutations are generated in the human BACs and the phenotype is analyzed in transgenic mice that are homozygous null mutants for the endogenous gene. These humanized mouse models provide an experimentally tractable system to generate mutations identified in humans and to analyze the mechanism by which they cause tumorigenesis. Functional analysis of BRCA1 and BRCA2 variants will also help us uncover any novel functions of these proteins. For example, analysis of a small 29-amino acid deletion in an evolutionarily conserved BRCA2 domain has revealed a role in alkyl-DNA repair. Embryonic fibroblasts expressing this mutant allele are sensitive to agents that alkylate guanine bases in the DNA. Repair of alkylated guanine primarily involves removal of the alkyl group by the O6-methylguanine-methyl transferase (MGMT) enzyme. This mutant has enabled us to uncover a role for BRCA2 in the repair of O6-mG adducts in addition to its function in RAD51-mediated DNA repair activity. We have also shown that BRCA2 associates with MGMT and the two proteins undergo degradation after alkylation. We have demonstrated that O6-benzylguanine (O6BG), a non-toxic inhibitor of MGMT, can also induce BRCA2 degradation. Because BRCA2 is a viable target for cancer therapy, our observation that O6BG induces degradation of BRCA2 may have significant clinical implications. Another aim of our research is to understand why a mutation in BRCA1 or BRCA2 results in predominantly breast and ovarian cancer although they are involved in DNA repair, a function that affects all cell types. Possibly, the response of the cells to damaged DNA is tissue specific, which could be a key factor in determining their fate. For example, some cells with damaged DNA may undergo cell death while others continue to proliferate and acquire additional mutations in genes involved in growth control or oncogenesis. To address this question, we have initiated an insertional mutagenesis approach in ES cells using Murine Stem Cell Virus (MSCV) to identify genes that genetically interact with BRCA1 or BRCA2. Because of our interest in DNA repair mechanisms, we have also undertaken the functional analysis of Rad51c, a member of the Rad51-like family of proteins involved in DNA repair. We have generated Rad51c mutant mice that reveal an early role of RAD51C in meiosis in males, marked by reduction of RAD51 foci at leptotene and persistence of DNA breaks at pachytene. In contrast, females display a defect in sister chromatid separation during metaphase II. These data provided the first in vivo evidence for a role for RAD51C during the late stages of meiotic recombination. Because of its essential role in DNA repair, we are now using this mouse model to examine the potential role of RAD51C as a tumor suppressor.